Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of Pi/PPi and SIBLING family genes/proteins during mineralization, highlighting the need for both physiochemical and cellular molecular factors to achieve homeostatic balance required for formation/regeneration of periodontal tissues. 1. Cell, Tissues and Animal Models of Periodontal Disease: a. Animal Models for regeneration of the periodontal apparatus: In previous studies, we have shown that factors controlling PPi/Pi levels such as ANK, ENPP1 and TNAP have significant roles during formation of the periodontium, especially cementum. In a proof of principle experiment using Ibsp KO mice, which exhibit marked periodontal tissue destruction due to limited cementum formation, we found that TNAP lentivirus, intramuscular delivery, rescued the Ibsp KO phenotype, e.g., insertion of PDL into newly formed cementum and increased alveolar bone volume. In a separate group of experiments periodontal fenestration defects were created in ANK and ENPP1 KO mice and 30 days later animals were sacrificed to evaluate wound healing. We noted increased cementum regeneration vs WT tissues. Based on this collective data, we believe strongly that factors that modulate PPi/Pi levels at local periodontal sites will promote cementogenesis. Next steps will be to deliver PPi regulators such as ALP and ENPP1 inhibitors directly at the sites of wound healing using the periodontal fenestration defect model and Ibsp KO mice. We have initiated a collaboration with Drs. William E. Bentley and Gregory Payne to develop ideal scaffolds for delivery of these factors. b. Ank, Enpp1 double KO mice and cells, in vitro: To determine the mechanisms by which ANK and ENPP1 regulate cementogenesis, e.g. identify whether their effects are additive/synergistic and if mechanistic differences exist in how they control cementogenesis, we generated Ank, Enpp1 dKO mice. Histological and microCT results to date indicate that acellular cementum phenotype exhibited by the Ank, Enpp1 dKO is a composite phenotype of the single KO (morphology and volume), suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Further, qPCR arrays conducted on RNA harvested from Ank and Enpp1 demonstrate unique gene expression profiles for Ank and Enpp1 single and double knockouts. Ongoing studies, using proteomics, qPCR microarray and RNA seq technologies, are focused toward defining protein and gene expression profiles of PDL tissues obtained from single and double KO mice at various stages of tooth root development. Further, as mentioned in 1a, we are contrasting periodontal wound healing capabilities of Ank vs Enpp1 KO mice. To complement in vivo studies, Ank KO, Enpp1KO and Ank, Enpp1 dKO cells are being generated using CRISPR-CAS technologies. We plan to perform RNA-seq on these cells, and studies using these cells will be paralleled with in vitro studies using siRNAs and ENPP1 inhibitors. c. Orthodontic tooth movement, using an Enpp1 KO mice model: Altered responses to orthodontic loading, decreased tooth movement, and altered osteoclast/odontoclast distribution in the Enpp1-/- mouse molars provide novel insights into the intersection of mineral metabolism and periodontal remodeling. Wolf et al JDR, 2018. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. 1. SIBLINGS: a. Spp1 (OPN) KO mice: Based on data to date, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. Foster et al. Bone 2018. b. Ibsp-KAE: In previous studies we reported Ibsp KO mice display a periodontal/cementum phenotype that resembles periodontal disease. As a step in defining the mechanism of BSP function in cementogenesis, we generated mice where the BSP RGD domain (integrin binding domain) was replaced by a non-functional KAE sequence (Ibsp-KAE). Results reveal that in contrast to Ibsp KO mice, Ibsp-KAE mice display normal cementogenesis (comparable to WT mice), but significantly increased osteoclasts along the alveolar bone similar to Ibsp KO mice. Despite the increased osteoclasts, micro CT analysis revealed a higher alveolar bone volume in older age Ibsp-KAE mice. In vitro studies focused on examining osteoclastogenesis potential of femur macrophages demonstrated normal maturation of cells obtained from Ibsp-KAE and WT mice, with severely defective maturation of cells obtained from Ibsp KO mice. Based on these results we hypothesize that Ibsp-KAE mice have a weakened PDL insertion into bone and cementum and that this triggers production of inflammatory cytokines that promote expression of RANKL and/or RANKL expression directly due to altered mechanical stress, resulting in increased osteoclast activity locally. Preliminary SEM data suggest weak insertion of PDL fibers into alveolar bone, however this needs to be confirmed. Ao et al. Bone 2017. Based on in vivo results we analyzed the migration and adhesion properties of Ibsp KO cementoblasts in vitro. Importantly, Ibsp KO cells migrated slower and exhibited decreased adhesion to tissue culture dishes when compared to WT cells providing evidence for weakened PDL properties we noted in vivo. We are in the process of preparing Ibsp-KAE cementoblasts using CRISPR-Cas technology in order to evaluate the role of the BSP RGD domain in modulating cell behavior during cementogenesis. c. BSP x OPN: In addition, we generated and are characterizing Spp1(OPN) x Ibsp(BSP) dKO mice to determine if loss of OPN expression would alter the periodontal phenotype reported for the Ibsp KO mice. Preliminary micro CT analysis indicate defective alveolar bone in dKO vs. WT and greater than that of Ibsp KO mice, suggesting additive, yet complex interactions between OPN and BSP in maintaining periodontal homeostasis. Histological analysis is ongoing. d. Collagen: Logic would suggest that individuals with osteogenesis imperfect (OI), a heritable disorder of the extracellular matrix characterized by low bone mass, brittle and fragile bones, and bone fractures, would have marked periodontal defects. We noted very minor changes in the periodontium using certain animal models and plan to submit a publication summarizing these data by Dec. 2018.